Molecular epidemiology of Blastocystis sp.

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Blastocystis sp. is the most common enteric protist of the human gastrointestinal tract. There has been continual controversy over the role Blastocystis plays in causing gastrointestinal disease in humans. It has been suggested to be a pathogen or an opportunistic commensal and it has also been suggested that pathogenicity could be related to subtype (ST) determined by molecular methods. Until recently there was little known about this protist in terms of epidemiology, pathogenicity and treatment. Clinical diagnosis has traditionally been based on microscopy of wet preparations or permanent stains but there has recently been a push towards more sensitive techniques such as culture and polymerase chain reaction (PCR). The correct diagnosis of Blastocystis is necessary for epidemiological and clinical studies which will aid in determining the actual role of this parasite in the gut and in producing disease. Due to the lack of knowledge on the pathogenicity of this parasite, research into treatment options is limited. Metronidazole is a commonly used anti-parasitic drug that has frequently been used for Blastocystis treatment. There is evidence that this drug may not actually have much efficacy at all on Blastocystis and therefore be the incorrect treatment option. This project was designed to address some of the shortcomings in the literature surrounding this parasite. The overall aim of the project was to describe the molecular epidemiology of Blastocystis sp. from Australia and comment on the pathogenicity of Blastocystis in humans. To be able to determine the molecular epidemiology, it was necessary to use the correct diagnostic method and therefore the first aim of this study was to determine the best diagnostic technique used for the detection of Blastocystis (aim 1 of this study). Five different techniques were tested for their sensitivity for detecting Blastocystis and it was found that microscopy of a permanent stain was the least sensitive at detecting Blastocystis and that PCR was the most sensitive technique. Once the most sensitive diagnostic technique was established it was then possible to determine the prevalence of Blastocystis within the Sydney population from clinical samples (aim 2 of this study). It was found that there was a 19% incidence of Blastocystis in this population and seven subtypes (STs) were identified by sequencing- ST1, ST2, ST3, ST4, ST6, ST7 and ST8. ST3 was found to be the most common ST in this population. This study then investigated the prevalence of Blastocystis in animals and determined the STs present (aim 3 of this study). There were 38 different species of animal from seven different locations investigated for the presence of Blastocystis using PCR. There were 80 (18%) positive isolates from 18 species, and nine different STs were identified- ST1, ST2, ST3, ST4, ST5, ST7, ST11, ST12 and ST13. This is the first report of Blastocystis from the eastern grey kangaroo, red kangaroo, wallaroo, snow leopard and ostrich. This study has expanded current knowledge on the host range of Blastocystis. Blastocystis is associated with symptoms in humans similar to irritable bowel syndrome (IBS) such as bloating, diarrhoea and abdominal pain and therefore this study aimed to look at the relationship between Blastocystis and IBS (aim 4 of this study). This study showed that though there was not a significantly higher percentage of Blastocystis seen in the IBS group compared to the control group, there was a difference in the STs present with ST4 only present in the IBS group. This study also highlighted the need for full microbiological work-up before a diagnosis of IBS can be given as Blastocystis, along with other microbes, may actually be a contributor to the disease process. The final part of this study was to look at treatment options for Blastocystis. Due to the lack of knowledge on the pathogenicity of Blastocystis there have only been a few studies on treatment options and much more information is needed (aim 5 of this study). This study followed 18 patients with chronic Blastocystis infection who were treated with a variety of antimicrobials. It was seen that the most common drug treatment of choice, metronidazole, was not effective for the clearance of Blastocystis. This study also highlighted the chronic nature of Blastocystis infection in the absence of any other infectious agents. This study also carried out in vitro testing for four common human Blastocystis STs ( ST1, ST3, ST4 and ST8) against 12 commonly used antimicrobials- metronidazole, paromomycin, ornidazole, albendazole, ivermectin, trimethoprim- sulfamethoxazole (TMP-SMX), furazolidone, nitazoxonide, secnidazole, fluconazole, nyastatin and itraconazole. Cultures were maintained in media that was determined the best for Blastocystis growth from aim 1 of this study. From this in vitro study the lack of efficacy of commonly used antimicrobials for the treatment of Blastocystis was shown in particular metronidazole, paromomycin and a triple therapy combination of furazolidone, nitazoxanide and secnidazole. This study did show the efficacy of two drugs- TMP-SMX and ivermectin and suggested the use of these treatments instead of metronidazole. Each of these studies aims has furthered the knowledge on Blastocystis epidemiology, pathogenicity and treatment options. This is the largest molecular epidemiological study to be completed in Australia and also the largest animal study to be undertaken thus far. Overall, this PhD project has contributed significantly by enhancing and extending current knowledge on Blastocystis and will hopefully encourage future research on this fascinating protist.
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